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Article

Plant Growth-Promoting and Tequila Vinasse-Resistant Bacterial Strains

by
Alberto J. Valencia-Botín
1,
Ismael F. Chávez-Díaz
2,
Florentina Zurita-Martínez
3,
Allan Tejeda-Ortega
3 and
Lily X. Zelaya-Molina
2,*
1
Plant Health Laboratory, Centro Universitario de la Cienega, University of Guadalajara, Ocotlán 47820, Jalisco, Mexico
2
Centro Nacional de Recursos Genéticos, Instituto Nacional de Investigaciones Forestales, Agricolas y Pecuarias, Tepatitlán de Morelos 47600, Jalisco, Mexico
3
Environmental Quality Research Center, Centro Universitario de la Cienega, University of Guadalajara, Ocotlán 47820, Jalisco, Mexico
*
Author to whom correspondence should be addressed.
Microbiol. Res. 2024, 15(3), 1144-1162; https://doi.org/10.3390/microbiolres15030077
Submission received: 4 June 2024 / Revised: 30 June 2024 / Accepted: 1 July 2024 / Published: 2 July 2024
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Abstract

:
Tequila vinasse, a byproduct of the tequila industry, is frequently discharged into water bodies or agricultural fields, posing significant ecological and human health risks. Bacterial communities that inhabit these agricultural fields have developed mechanisms to utilize tequila vinasse as a potential nutrient source and to promote plant growth. In this study, strains from the phyla Actinomycetota and Pseudomonadota were isolated from agricultural fields irrigated with tequila vinasse for 2, 10, and 14 years in Jalisco, Mexico. The results showed that strains of Terrabacter, Azotobacter, Agromyces, Prescottella, and Leifsonia tolerate high concentrations of tequila vinasse and promote maize seedling growth in the presence of tequila vinasse. Additionally, some of the strains solubilize potassium and produce siderophores, cellulase, protease, lipase, and esterase. The strains Terrabacter sp. WCNS1C, Azotobacter sp. WCNS1D, and Azotobacter sp. WCNS2A have potential applications in the bioremediation of tequila vinasse in agricultural fields discharged with tequila vinasse.

1. Introduction

During the production of tequila, vinasse is produced as a dark-colored byproduct [1]. For every liter of tequila produced, approximately 10 to 15 L of liquid vinasse are generated, posing waste management challenges for distilleries and regulatory bodies [2]. Despite federal regulations, tequila vinasse is often discharged into agricultural fields or water bodies without proper treatment, leading to significant environmental contamination. Tequila vinasse is considered a hazardous substance for ecosystems, including soils exposed to these discharges. Tequila vinasse typically contains residual sugars, alcohol, and various non-volatile compounds. Physicochemically, tequila vinasse is characterized by an acidic pH (3.9–5.1), high chemical oxygen demand (50,000–95,000 mg∙L−1), high biological oxygen demand (18,900–37,000 mg∙L−1), high total volatile solids (up to 82,222 mg∙L−1), and high potassium content (10–345 g∙L−1) [3]. Vinasse generation is not exclusive to tequila factories, as it is also generated in the production of other alcoholic beverages, such as mezcal, as well as in ethanol production.
Several alternatives have been developed to recycle vinasses, i.e., fertirrigation, fermentation, energy production, yeast production, animal feed production, and concentration by evaporation [4]. Sugarcane vinasse is one of the most studied byproducts due to its generation volume in bioethanol production; it is mainly employed in fertilization and irrigation practices, but impactful technologies such as microbial fermentation, anaerobic digestion, and oxidation and chemical processes have been developed to reduce its organic and mineral load, as well as to transform its organic matter into nutritious biomass [5]. Some studies have focused on treating and adding value to tequila vinasse; for example, Cea-Barcia et al. [1] developed a microalgae–yeast biomass-based technology of interest to aquaculture, while Díaz-Vázquez et al. [6] proposed a fermentation process of tequila vinasse, by a mixed culture of fodder yeast species, to produce protein feed to livestock industry.
Vinasse has been utilized as a fertilizer for its advantageous properties such as high calcium and potassium content. However, some vinasses may also contain salts, metals, and dissolved solids, posing potential risks of soil and water contamination [3,7]. Additionally, the application of vinasses to agricultural fields affects the microbial populations involved in the biological process of decomposition of organic matter, nitrification, denitrification, and fixation of N2, but also facilitates the action of microorganisms that improve soil structure [8,9,10,11]. Santos et al. [12] reported that sugarcane vinasse altered the population of fungi and bacteria in the irrigated fields, mainly actinomycetes and cellulolytic bacteria, while Camargo [13] reported that vinasses specifically increased the fungal genera of Neurospora, Aspergillus, Penicillium, Mucor, and the bacterial genus Streptomyces. In addition, Tejada et al. [14] reported that beet vinasse interfered with soil microbe biomass, respiration, and enzymatic activities.
The use of microbial communities to bioremediate tequila vinasses is not a common practice; some studies have focused on using yeast species for tequila vinasse fermentation, but there are no studies of plant growth-promoting bacteria from tequila vinasse-irrigated agricultural fields that assimilate the vinasse to support their growth and plant growth. For this reason, this study aimed to isolate plant growth-promoting and tequila-vinasse resistant bacterial strains from agricultural fields irrigated with vinasse for two, ten, and fourteen years, to contribute to the bioremediation efforts of tequila vinasse in agricultural fields. Bacterial strains were phylogenetically identified and evaluated under in vitro conditions to determine their potential resistance to vinasse and their plant growth-promoting capacities in the presence of tequila vinasse, traits that could contribute to their adaptation to soils irrigated with vinasse. This study could be useful in developing bioremediation technologies to treat vinasse.

2. Materials and Methods

2.1. Isolation of Bacterial Strains

Soil samples were collected from three plots in the State of Jalisco, Mexico. Two plots were located in the Capilla de Guadalupe locality, Tepatitlán de Morelos municipality, at 20°50′14.7″ N 102°34′56.8″ W (S1) and at 20°50′14.6″ N 102°34′56.6″ W (S2); and a plot was situated in a tequila-producing micro-enterprise in the Tototlán municipality, at 20°32′25.8″ N 102°38′21.4″ W (S3). The three plots varied in frequency and duration of tequila vinasse discharge. S1 was irrigated daily with vinasse for one month per year over two years; S2 received daily irrigation for two months per year for ten years; and S3 was watered daily for one month, three to four months per year over fourteen years. Additionally, S1 and S3 produced wild grass for livestock consumption, while S2 was dedicated to maize cultivation.
Serial tenfold dilutions of up to 10−4 from 1 g of the samples of tequila vinasse-irrigated soils were inoculated into Winogradsky’s N-Free Medium [15] without sugar solution and amended with 2% of previously neutralized tequila vinasses. All the plates were incubated at 26 °C for 3–5 days. Bacterial colonies with different morphotypes were described morphologically, counted as colony forming units (CFU g−1 of vinasses irrigated soil), and plated on modified Winogradsky medium to obtain pure cultures. All the strains were conserved at −80 °C in 30% glycerol.
Serial tenfold dilutions up to 10−4 from 1 g of the samples of tequila vinasse-irrigated soils were inoculated into modified Winogradsky’s N-Free Medium [15] without sugar solution and amended with 2% of previously neutralized tequila vinasses. All the plates were incubated at 26 °C for 3–5 days. Bacterial colonies were grouped into morphotypes, and each morphotype was described morphologically. The colonies were counted as colony-forming units (CFU) per gram of vinasse-irrigated soil and plated on modified Winogradsky medium to obtain pure cultures. All the strains were conserved at −80 °C in 30% glycerol.

2.2. Identification of Bacterial Strains

The strains were identified at the genus level through phylogenetic analysis of a partial sequence of the 16S rRNA gene. Amplification and sequencing of the 16S rRNA gene were conducted by Macrogen Inc. (Seoul, Republic of Korea) using the default service, with the 27f/1493r and 785F/907R universal primers for amplification and sequencing, respectively. To generate a consensus 16S rDNA sequence for each strain, the sequences obtained with the 785F and 907R primers were edited and overlapped using BioEdit 7.2.5 software [16]. For each strain, a collection of taxonomically related sequences of type strains was obtained from GenBank to construct the phylogenetic tree. Sequence alignment, selection of nucleotide substitution model, and phylogenetic analyses were performed using MEGA version 7 [17]. The phylogenetic tree was constructed with a bootstrap of 1000 replications, utilizing the maximum likelihood method and the Tamura–Nei substitution model. All sequences generated in this study were deposited in GenBank under the accession numbers OM060440–OM060458.

2.3. Plant Growth Promotion and Biocontrol Traits of the Bacterial Strains

From pure bacterial cultures grown on TSA for 24 h at 25 °C, 10 μL of the suspensions, adjusted to a McFarland nephelometer 0.5 tube, were inoculated into Aleksandrow [18], NBRIP [19], Zn–Tris-minimal [20], CAS [21], Congo Red [22], Tween 80-Agar, Tween 20-Agar [23], Castañeda–pectin, Castañeda–starch, and skim-milk media [24] to assess the strains’ capacity to solubilize K, PO4, and Zn, and to produce siderophore, cellulase, esterase, lipase, pectinase, amylase, and protease, respectively. Solubilization of K was indicated by a yellow halo around the bacterial colonies grown on the specific media. Solubilization of PO4, and Zn was indicated by a transparent halo around the colonies grown on the respective media. Siderophore production was identified by a yellow-orange halo around the colonies on CAS medium, while cellulase production was identified by a yellowish halo surrounding colonies on Congo-red medium. For amylase and pectinase production, a clear halo around colonies flooded in a 1% w/v iodine solution and in 5% w/v cetyltrimethylammonium bromide (CTAB), respectively, was considered a positive result. Esterase and lipase activity were demonstrated by the formation of a halo of crystals around the colonies on the respective media. All Petri plates were incubated at 25 °C for 1–3 days.

2.4. Tequila Vinasses’ Minimal Inhibitory Concentration (MIC) on Bacterial Strains

For each bacterial strain, 5 μL of a suspension at 1 × 106 CFU mL−1 was spotted on Winogradsky N-free medium without sugar solution and then supplemented with neutralized tequila vinasses, ranging from 0 to 30%, in 13 treatments with increments of 2.5%. Another set of 13 treatments was established under the same conditions and supplemented with 1% dextrose. All Petri dishes were incubated at 26 °C and examined for growth after 3, 6, and 9 days. Growth records on day 9 were considered as the minimal inhibitory concentrations of tequila vinasses (TV-MICs).

2.5. Plant-Growth Promotion by Bacterial Strains

The modified ISTA germination method [25] was utilized to assess the plant growth-promoting capacities of the bacterial strains on maize seeds of the Agroferza H-311 hybrid (Agroferza S.A. de CV. seed company, Zacatecas, Mexico). Briefly, 100 seeds underwent washing and disinfection in chlorine gas (3.5 mL of HCl 12 N in 100 mL of NaClO 5.25%) for 6 h, followed by rinsing in sterile distilled water. Subsequently, 25 seeds were arranged in five alternate rows between sterile cellulose paper (Thermal paper; Sarasota, FL, USA), onto which 15 mL of a bacterial suspension at 1 × 106 CFU mL−1 were applied. The paper containing the seeds was then rolled up, and the rolls were incubated at 25 °C in a growth chamber (Binder KBWF 720, Tuttlingen, Germany) under a photoperiod of 14 h light/10 h dark, with 75% humidity. Each treatment was replicated four times, and four rolls supplemented with 15 mL of sterile distilled water served as the control. After 12 days, the measured parameters included the plumule length (PL), seedling root length (RL), seedling length (SL), number of roots (NR), plumule fresh weight (PFW), plumule dry weight (PDW), roots fresh weight (RFW), roots dry weight (RDW), seedling fresh weight (SFW), and seedling dry weight (SDW). The data were subjected to an analysis of variance (ANOVA) and Tukey’s mean separation test (p ≤ 0.05), which were performed in Minitab® 17 statistical software. Additionally, Principal Component Analysis (PCA) was conducted using Minitab® 17 software to ascertain the differences among the various treatments involving the bacterial strains.

2.6. Effect of Soils Irrigated with Vinasse on Plant Growth

The effect of tequila vinasse irrigation on plant growth in three different soils (S1, S2, S3) was evaluated using a maize seed germination test with P3011W maize hybrid seeds (Pioneer, Johnston, IA, USA). Germination trays, each containing 100 cells, were filled with 15 g of soil and one seed per cell. Garden soil (SC) served as the control. The experiment was replicated three times. Initially, trays were thoroughly saturated during watering, followed by the addition of 2 mL of water per cell daily for 18 days. Distilled water was used for irrigation. Upon completion of the evaluation period, the maize germination rate was determined using the following formula: Germination rate (%) = (Number of germinated seeds/Total number of seeds) × 100% [26].

2.7. Plant Growth Promotion by Bacterial Strains in the Presence of Tequila Vinasse

To evaluate the plant growth-promoting capacities of the bacterial strains in the presence of tequila vinasse, a germination test was conducted using seeds of the maize hybrid Agroferza H-311. The test followed the previously outlined conditions, with the addition of 7.5 mL of a bacterial suspension at 2 × 106 CFU mL−1 and 7.5 mL of 2.5% tequila vinasse to each paper roll. For the blank treatments, four paper rolls were added with 15 mL of the bacterial suspensions at 1 × 106 CFU mL−1. Additionally, four paper rolls were added with 7.5 mL of 2.5% tequila vinasse and 7.5 mL of sterile distilled water as the positive control, while four paper rolls were added with 15 mL of sterile distilled water as the negative control. After 12 days, the same parameters mentioned previously were evaluated. The collected data underwent a two-way ANOVA and Tukey’s mean separation test (p ≤ 0.05), and both analyses were conducted using the Minitab® 17 software. Additionally, PCA was performed in Minitab® 17 to discern the differences among the various treatments concerning the effect of the bacterial strains and tequila vinasse on maize seedlings.

3. Results

3.1. Selection and Identification of Bacterial Strains

A total of 19 bacterial morphotypes were obtained from the three soil samples (Table 1); the morphotypes varied in size (1–3 mm), shape (circular/irregular), color (white/beige), and margin (entire/lobate). Analysis of the 16S rRNA gene identified the strains as members of 11 different genera from two bacterial phyla (Table 2). These included the genera Agromyces, Arthrobacter, Leifsonia, Microbacterium, Prescottella, Rhodococcus, Sinomonas, Streptomyces, and Terrabacter from the phylum Actinomycetota, as well as Azotobacter and Pseudomonas from the phylum Pseudomonadota (Figure 1). The sequences of the 16S rRNA of Agromyces sp. WCNS1A and WCNS1F had 100% identity between them, while the sequences of Arthrobacter sp. WCNS2C and WCNS2E had 94.5% identity. Similarly, the sequences of Azotobacter sp. WCNS1D, WCNS2A, WCNS2B, WCNS3B, WCNS3D and WCNS3F had 99.9–100% identity among them, and the sequences of Rhodococcus sp. WCNS3A and WCNS3C had 100% identity. Additionally, the sequences of the bacterial strains obtained in this study had 97.4–100% identity with the sequence of the type strains most closely related phylogenetically (Table 2).
Azotobacter was the most abundant genus, with seven strains obtained from the three soil samples, primarily from the sample where vinasses were continuously applied for one month, three to four times a year, over 14 years (Table 1). In the modified N-free Winogradsky, Azotobacter strains exhibited colonies that were beige, soft, flat, and shiny, ranging from translucent to opaque, and from 0.5 to 2 mm. Azotobacter population levels in the samples varied between 104 and 105 CFU g−1 (Table 1).

3.2. Plant Growth Promotion and Biocontrol Capacity of Bacterial Strains

The 19 bacterial strains were evaluated for their ability to solubilize K, Zn, and PO4, as well as for their production of siderophore, amylase, cellulase, protease, pectinase, lipase, and esterase in specific media. The results showed that Pseudomonas sp. WCNS1F solubilized K, while Agromyces sp. WCNS1E solubilized both K and PO4 (Table 3). Pseudomonas sp. WCNS1F, Agromyces sp. WCNS1E, Leifsonia sp. WCNS2G, Rhodococcus sp. WCNS3A and WCNS3C, and Azotobacter sp. WCNS3E produced siderophores (Table 3). Agromyces sp. WCNS1A and WCNS1E, Microbacterium sp. WCNS1B, Terrabacter sp. WCNS1C, and Arthrobacter sp. WCNS2E produced cellulase. Pseudomonas sp. WCNS1F, Rhodococcus sp. WCNS3A, and WCNS3C produced esterase. Additionally, Agromyces sp. WCNS1A and Terrabacter sp. WCNS1C produced protease, while Pseudomonas sp. WCNS1F and Sinomonas sp. WCNS2C produced lipase. Only Rhodococcus sp. WCNS3C produced amylase. The strains Prescottella sp. WCNS2D, Streptomyces sp. WCNS2F, and Azotobacter sp. WCNS1D, WCNS2A, WCNS2B, WCNS3B, WCNS3D, and WCNS3F showed no activity. None of the strains were able to solubilize Zn or produce pectinase. Pseudomonas sp. WCNS1F and Agromyces sp. WCNS1E exhibited the greatest number of activities. Additionally, Agromyces sp. WCNS1A exhibited the largest halos for protease (0.96 cm) and cellulase (1.82 cm) production, Pseudomonas sp. WCNS1F for lipase (1.23 cm) and esterase (1.05 cm) production, and Agromyces sp. WCNS1E for cellulase (1.37 cm) production (Table 3).

3.3. Tequila Vinasses’ Minimal Inhibitory Concentrations (MICs)

The tolerance of the bacterial strains to tequila vinasse varied widely among the strains in the assay media. The MICs of the strains were variable, ranging from 12.5 to >30% and from 7.5 to >30% of the vinasses in the Winogradsky N-free medium with and without sugar solution, respectively (Table 1). Azotobacter sp. WCNS2A isolated from soil sample S2 exhibited the highest tolerance to tequila vinasse, with a MIC of 30% in both media. Similarly, strain Prescottella sp. WCNS2D from soil sample S2 showed MICs of 25% and >30% in the Winogradsky medium with and without dextrose, respectively (Table 1). Furthermore, the strains Terrabacter sp. WCNS1C and Azotobacter sp. WCNS1D, from soil sample S1, displayed MICs of >30% in the Winogradsky medium without dextrose, while the strain Pseudomonas sp. WCNS1E had a MIC of 25% in the Winogradsky medium with dextrose. From soil sample S3, the strain Rhodococcus sp. WCNS3A also had a MIC of >30% in medium without dextrose, and the strains Azotobacter sp. WCNS3B and WCNS3E demonstrated a MIC of >30% of vinasses in the Winogradsky medium with dextrose (Table 1).

3.4. Plant Growth-Promoting Bioassay

Sixteen bacterial strains were selected to carry out the plant growth-promoting evaluation on seed germination of H-311 maize hybrid. After twelve days of the experiment setup, a mixed effect of bacterial strains on maize seedling development was observed (Figure 2).
Almost all the strains generated an increase in PL, RL, SL, NR, RFW, RDW, PFW, PDW, SFW, and SDW (Table 4). Additionally, a significant positive effect was quantified in some strains in the different measured parameters of PL (4 strains), NR (1 strain), NR (3 strains), RFW (11 strains), RDW (6 strains), PFW (7 strains), SFW (7 strains) and SDW (1 strain) (Table 4). Therefore, the greatest positive effect of the bacterial strains was observed in the variables of RFW, RDW, PFW, and SFW. In addition, a significant negative effect was quantified in some strains in the variables of RL (2 strains), SL (1 strain), PFW (3 strains), and SFW (1 strain) (Table 4).
Considering the effect of each bacterial strain on the growth of the seedlings, Agromyces sp. WCNS1A, Terrabacter sp. WCNS1C, Agromyces sp. WCNS1F, Azotobacter sp. WCNS2A, Leifsonia sp. WCNS2G, and Azotobacter sp. WCNS3B exhibited a significant increase in 4–6 of the 10 evaluated variables (Table 4). However, the PCA indicated that Leifsonia sp. WCNS2G is the most promising plant growth-promoting strain; followed by the strains Agromyces sp. WCNS1F, Azotobacter sp. WCNS2A, Rhodococcus sp. WCNS3A, and Azotobacter sp. WCNS3B (Figure 3). This result is supported by the fact that the first two principal components account for 65.1% of the total variation, and the parameters contributing to this variation include PDW, PFW, SFW, SDW, NR, and PL. Furthermore, the five strains had an increase of 17.5–32.7% in PL, 5.0–20.2% in RL, 3.2–18.0% in SL, 10.5–68.4% in NR, 21.9–50.1% in RFW, 6.4–44.6% in RDW, 14.15–33.0% in PFW, 4.55–23.4% in PDW, 15.9–34.9% in SFW, and 5.6–26.5% in SDW, and a decrease of 17.2% in RL.

3.5. Maize Germination on Soils Irrigated with Vinasse

The effect of soils irrigated with tequila vinasse (S1, S2, S3) on plant growth was assessed through the germination of P3011W maize hybrid seeds (Pioneer) from day 4 to day 20 after sowing (Figure 4).
Maize seed germination and vigor increased in S1, S2, and S3, in comparison with the control. In S1, seed germination increased by 50% during the evaluation period of 20 days and reached the total germination level of the control (58 seeds) in approximately a quarter of the time (5 days) (Figure 4). While in S2, seed germination increased by 17% over the evaluation period and reached the total germination level of the control in almost half the time (9 days). Finally, in S3, seed germination increased by 33% during the evaluation period, and reached the total germination level of the control (58 seeds) also in almost half the time (8 days) (Figure 4).

3.6. Plant Growth-Promoting Bioassay under Tequila Vinasse and Bacterial Strain Influence

The bioassay evaluated the effect of 1.25% vinasse and bacterial strains on germination and seedling development of the maize hybrid H-311, using the following bacterial strains: Terrabacter sp. WCNS1C, Azotobacter sp. WCNS1D, Agromyces sp. WCNS1F, Azotobacter sp. WCNS2A, Prescottella sp. WCNS2D, Leifsonia sp. WCNS2G, Rhodococcus sp. WCNS3A, Rhodococcus sp. WCNS3C, and Azotobacter sp. WCNS3E. After twelve days, the application of vinasse and bacterial strains generated a significant increase in PL, RL, SL, RFW, RDW, PFW, PDW, SFW, and SDW, in comparison with the controls (Figure 5).
Specifically, the bacterial strains combined with vinasse generated greater results, with an increase of 17.3–39.0% in PL, 32.0–53.5% in RL, 27.3–45.9% in SL, 27.8–65.7% in NR, 56.5–132.3% in RFW, 20.0–50.0% in RDW, 47.2–104.9% in PFW, 20.2–54.8% in PDW, 54.4–101.8% in SFW, and 22.8–53.7% in SDW (Table 5), in comparison with the negative control (without both vinasse and bacteria). In comparison with the positive control (with vinasse but without bacteria), the bacterial strains together with vinasse generated an increase of 10.9–31.5% in PL, 13.2–30.5% in RL, 14.7–31.5% in SL, 14.0–47.8% in NR, 44.1–89.5% in RFW, 17.1–46.3% in RDW, 30.7–82.0% in PFW, 13.8–46.4% in PDW, 35.9–77.5% in SFW, and 16.8–46.2% in SDW (Table 5).
Additionally, the effect of vinasse was obtained comparing the treatment of each bacterial strain with its blank (without vinasse); in this case, the addition of 1.25% vinasse increased PL by 1.2–29.7%, RL by 2.3–35.0%, SL by 3.9–24.4%, NR by 10.7–53.3%, RFW by 7.1–77.7%, RDW by 5.9–40.5%, PFW by 10.9–68.6%, PDW by 5.0–44.9%, SFW by 11.4–69.8%, and SDW by 5.2–44.2%. Furthermore, the effect of the bacterial strains was obtained by comparing the blank of each strain with the negative control. In this way, the bacterial strains increased PL by 7.2–21.4%, RL by 2.6–36.1%, SL by 6.9–28.4%, NR by 12.1–41.3%, RFW by 33.1–82.3%, RDW by 10.0–27.5%, PPFW by 6.9–77.0%, PDW by 4.1–20.2%, SFW by 9.0–75.2%, and SDW by 2.1–22.3% (Table 5).
Moreover, a significant positive effect was quantified in the different measured parameters as a result of the treatments of the join effect of the vinasse and bacteria (Table 5). Vinasse, together with each of the nine bacterial strains, generated a significant increase in the variables of PL, RL, SL, RFW, PFW, PDW, SFW, and SDW (Table 5). The PCA clearly shows the effect of the bacterial strains, with and without vinasse, on maize seedling growth (Figure 6). Specifically, the PCA showed that the strains Terrabacter sp. WCNS1C, Azotobacter sp. WCNS1D, and Azotobacter sp. WCNS2A, supplemented with 1.25% vinasse, generated the greatest increases in maize seedlings development (Figure 6). This result is based on the first principal component, which explains 78.0% of the total variation, considering all the evaluated parameters. Also, these strains had an increase of 12.7–15.9% in LP, 2.3–19.1% in LR, 9.0–16.1% in LS, 17.2–23.0% in NR, 7.1–77.7% in PFR, 1.2–40.5% in PSR, 16.6–68.6% in PFP, 23.0–44.9% in PSP, 20.8–69.8% in SFW, and 18.7–44.2% in SDW.

4. Discussion

Tequila is a traditional Mexican fermented alcoholic beverage made from Agave tequilana Weber var. blue. Although its origins date back to the late 16th century [27], it has become a growing industry today, with a production of 598.7 million liters in 2023 [28]. Over the last 20 years, its consumption has increased by 453% (2281 thousand tons in 2023) and its exportation has risen by 295% (401 million liters in 2023) [28]. Additionally, it has directly or indirectly generated 300,000 jobs [29]. Tequila has the Tequila Denomination of Origin seal, which protects its production in 181 municipalities located in five Mexican states. Moreover, tequila production, tourism, and commercial representation offices are concentrated in the state of Jalisco [29], where significant efforts are underway to develop alternatives for recycling tequila vinasse and reducing the environmental pollution caused by this byproduct.
The main application of vinasses worldwide has been in agricultural fields, primarily through fertirrigation, as it represents a low investment and replaces the use of chemical fertilizers, particularly those containing phosphorus [30]. Sugarcane vinasse has been applied in fertirrigation in Brazil since the 1950s; currently, 75–80% of the area planted with sugarcane can be irrigated with vinasse [30]. However, the direct application of vinasse to the soil can cause salinization, leaching of metals, changes in soil quality, alkalinity reduction, crop losses, increase in phytotoxicity, and unpleasant odor. Therefore, fertirrigation may be a palliative practice that gives a false impression of efficiently solving the problem of vinasse disposal [31,32]. The use of tequila vinasse in soil is a widespread practice in Mexico. However, there are no documented studies on the potential impact of vinasse application on soil quality. It is possible that both adverse and beneficial effects could occur, as has been reported with other types of vinasses [3]. Zurita et al. [2] reported that in some regions of Jalisco, tequila micro-factories generally do not treat their vinasse and mostly discharge it directly onto soils. However, soil fertility has not been affected due to a resting period for the soil after the vinasse has been discharged and before planting crops. This study isolated bacterial strains from those agriculture fields discharged with vinasse for two, ten, and fourteen years to determine their plant growth-promoting capacities and resistance to soils irrigated with vinasse.
In this study, 19 bacterial strains from 11 genera were isolated from three vinasse-irrigated soils. These strains are plant growth-promoting bacteria with the ability to either use tequila vinasse as a carbon and nitrogen source or fix nitrogen and carbon from the environment while degrading tequila vinasse. The soil with the longest record of vinasse discharge (S3) was consistently inhabited by strains from the genera Azotobacter and Rhodococcus, whereas a greater diversity of bacteria from the Actinomycetes class predominated in S1 and S2. In general, these bacterial strains can grow in an environment with high calcium and potassium content, and tolerate salts, heavy metals, and dissolved solids [3,7]. Additionally, the physiological, biochemical, and enzyme activities of the bacterial strains varied. Some strains from all three soil samples produced siderophores, while strains exclusively from S1 solubilized K and/or PO4, and produced cellulase and protease, and strains predominantly from S3 produced esterase. Strains from both S1 and S2 produced lipase. Notably, strains from the S1 soil sample demonstrated the highest number of activities and exhibited the largest halos of hydrolytic enzyme production.
The strains from the genera Agromyces, Azotobacter, Leifsonia, Prescottella, Rhodococcus, and Terrabacter were the most significant due to their ability to promote plant growth in the presence of tequila vinasse. Strains of the genus Agromyces have been isolated from heavy metal-polluted environments, where they play roles in leaching, accumulating, or immobilizing heavy metals [33,34,35]. They are also found as part of the microbial communities that degrade agricultural residues [36] and have been reported as PGPR inhabiting the rhizosphere of some crops [37]. The strain Agromyces sp. WCNS1F isolated in this study produced lipases, esterases, and siderophores; additionally, this strain can grow on Winogradsky media amended with tequila vinasse and promote maize seedlings growth, particularly in root parameters. The strain WCNS1F is related to Agromyces fucosus, and strains of this species isolated from soil and plants have been reported to metabolize organophosphonate and phosphonoacetate compounds as phosphorus and carbon sources, respectively, and to use aromatic and aliphatic hydrocarbons as carbon sources [38,39].
Strains of Azotobacter were predominantly found in all the studied soils. Strains of this genus are well-known PGPR for their ability to fix nitrogen, improve shoot and root development, suppress pathogenic bacteria and fungi, increase P concentration, and produce metabolites such as amino acids [40]. Although it has been reported that Azotobacter species are sensitive to acidic pH, high salt concentration, and temperature [41], the Azotobacter strains in this study grew in soils irrigated with tequila vinasse. Azotobacter strains utilize a broad spectrum of substrates, including organic acids, aromatic compounds, hydrocarbons, phenolic compounds, herbicides, and insecticides as sources of carbon and energy, forming several metabolites [42,43,44,45], and they are resistant to heavy metals [46]. Therefore, they can participate in the bioremediation of polluted environments. For example, strains of Azotobacter are PGPR for groundnut in the Hg-contaminated mine tailing [47] and participate in the bioremediation of oil-contaminated environments [48]. Specifically, Azotobacter strains have been reported to produce exopolysaccharides and biopolymers from sugarcane vinasse [49,50]. Azotobacter sp. WCNS1D, WCNS2A, and WCNS3E did not exhibit many plant growth-promoting capacities under in vitro tests, but they exhibited a MIC of 17≥30% in the Winogradsky–tequila vinasse medium and promoted maize seedling growth with tequila vinasse, mainly in terms of plumule and root length, number of roots, and fresh and dry weight of the plumule; these strains are closely related to Azotobacter bryophylli, a nitrogen-fixing strain isolated from leaves of Bryophyllum pinnatum [51].
Strains of Terrabacter have been reported to metabolize DDE, a residue of DDT [52], hydrochars, herbicides [53], dioxins [54], cotinine [55], and aromatic compounds [56]. They are also known as rhizospheric strains with heavy metal resistance [57], and as the PGPR of crops such as sugarcane [58]. Terrabacter sp. WCNS1C is related to Terrabacter terrae and Terrabacter tumescens. T. terrae, isolated from soil mixed with Iberian pig hair, shows keratinase activity and can grow using a variety of carbohydrates as carbon source [59], and it can also degrade dalapon [60]. T. tumescens has been reported to biomineralize heavy metals [61]. The strain Terrabacter sp. WCNS1C produced protease and cellulase and had a MIC of 12.5% and >30% in the Winogradsky–tequila vinasse medium with and without dextrose, respectively. This strain promoted the growth of maize seedlings with tequila vinasse, particularly in the number of roots, and the length, fresh weight, and dry weight of the plumules. Terrabacter sp. WCNS1C has the potential to be used in bioremediation of tequila vinasse in agricultural fields.
Leifsonia sp. WCNS2G promoted the growth of maize seedlings in the presence of tequila vinasse, particularly enhancing the number of roots, and the length, fresh weight, and dry weight of plumules. The strain produced siderophores and had a MIC of 22.5 and 7.5% in the Winogradsky–tequila vinasse medium with and without dextrose, respectively. Leifsonia strains have been reported to modulate several responses for biotic and abiotic stress tolerance [62]. For example, they function as PGPR for maize [63], while Aquilegia viridiflora [64] acts as root endophytes with heavy metal resistance in the Ni-hyperaccumulator, Alyssum bertolonii [65], and produce biofuel [66]. Leifsonia sp. WCNS2G is closely related to Leifsonia xyli and Leifsonia shinshuensis; although the two subspecies of L. xyli, L. xyli subsp. xyli and L. xyli subsp. cynodontis, have been reported as plant pathogens of sugarcane and Bermudagrass [67], respectively, they have also been reported as PGPR that mitigates the stress caused by copper in tomato plants [68]. Moreover, L. shinshuensis can metabolize molecules such as carbamazepine and xylene [69,70].
Prescottella sp. WCNS2D had a MIC of 25 and 30% in Winogradsky–tequila vinasse medium with and without dextrose, respectively. Additionally, the strain promoted the growth of maize seedlings in the presence of vinasse, particularly increasing the number and the dry weight of the roots, as well as the plumules. Prescottella strains have been reported to inhabit extreme environments, degrading pollutants, pharmaceutical compounds [71,72], phenolic compounds [73], and petroleum hydrocarbons [74]. They are essential in environmental biodegradation [75] and bioremediation [76]. Prescottella sp. WCNS2D is closely related to Prescottella equi and Prescottella soli. Although P. equi has been associated with human and animal diseases [75], some strains have a high plastic-degrading potential [77]. P. soli isolated from soil [78] has been reported as a glyphosate-degrading species [79].
Additionally, strains of Arthrobacter, Microbacterium, Pseudomonas, Sinomonas, Streptomyces, and Rhodococcus have been reported from a wide variety of environments, including extreme conditions such as low temperatures, industrial residues, agrochemicals, heavy metals, and more. These strains have the capacity to degrade a wide range of natural organic and xenobiotic compounds and have valuable applications in polluted environments [80,81,82,83]. All the strains in this study are resistant to tequila vinasse-irrigated soil and play key roles in the geological cycles of carbon, nitrogen, phosphorus, potassium, iron, and sulfur, helping to restore soil fertility in these environments [84]. Some of the genera found in these soils have been reported in both crop and non-crop soils, but none specifically from soils irrigated with tequila vinasse. These strains have potential applications in bioremediation not only of vinasse, but also of heavy metals, natural organic, and xenobiotic compounds. However, further experiments are required to define potential future applications.

5. Conclusions

The strains from the genera Azotobacter and Terrabacter, isolated from agricultural soils irrigated with tequila vinasse, exhibit phenotypical traits for plant growth promotion and tequila vinasse tolerance. Specifically, the strains Terrabacter sp. WCNS1C, Azotobacter sp. WCNS1D, and Azotobacter sp. WCNS2A can grow under high concentrations of tequila vinasse and promote the growth of maize seedling. These strains have the potential to be used in applications in the bioremediation of tequila vinasse in agricultural fields.

Author Contributions

Conceptualization, A.J.V.-B., I.F.C.-D. and L.X.Z.-M.; methodology, A.J.V.-B., I.F.C.-D., F.Z.-M., A.T.-O. and L.X.Z.-M.; software, A.J.V.-B. and L.X.Z.-M.; validation, A.J.V.-B. and L.X.Z.-M.; formal analysis A.J.V.-B. and L.X.Z.-M.; investigation, A.J.V.-B., I.F.C.-D. and L.X.Z.-M.; resources, A.J.V.-B. and L.X.Z.-M.; data curation, A.J.V.-B. and L.X.Z.-M.; writing—original draft preparation, A.J.V.-B. and L.X.Z.-M.; writing—review and editing, A.J.V.-B., I.F.C.-D., F.Z.-M., A.T.-O. and L.X.Z.-M.; visualization, A.J.V.-B. and L.X.Z.-M.; supervision, A.J.V.-B. and L.X.Z.-M.; project administration, A.J.V.-B. and F.Z.-M.; funding acquisition, A.J.V.-B., F.Z.-M. and L.X.Z.-M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded partially by “FONDO SECTORIAL DE INVESTIGACIÓN Y DESARROLLO SOBRE EL AGUA, grant number A3-S-66470”.

Data Availability Statement

All the sequences obtained in this study were deposited at GenBank under the accession numbers: OM060440-OM060458. All data will be made available on request.

Acknowledgments

The authors sincerely thank Geovanna L. Ortíz, Yaret Gallegos-Remedios-Rodríguez, and Zoe Resendiz-Venado for their technical support.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Phylogenetic tree of partial sequences the 16S rRNA gene of bacterial strains isolated from tequila vinasse-irrigated soils, inferred with the maximum likelihood phylogenetic tree based on the Tamura–Nei substitution model. The numbers at the nodes indicate bootstrap values of 1000 replicates. Branch lengths are proportional to the number of substitutions per site (see scale bars).
Figure 1. Phylogenetic tree of partial sequences the 16S rRNA gene of bacterial strains isolated from tequila vinasse-irrigated soils, inferred with the maximum likelihood phylogenetic tree based on the Tamura–Nei substitution model. The numbers at the nodes indicate bootstrap values of 1000 replicates. Branch lengths are proportional to the number of substitutions per site (see scale bars).
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Figure 2. Germination bioassay using H-311 maize hybrid seeds inoculated with: (a) Control, (b) Leifsonia sp. WCNS2G, (c) Agromyces sp. WCNS1F, (d) Rhodococcus sp. WCNS3A (e) Terrabacter sp. WCNS1C, (f), Sinomonas sp. WCNS2C, (g) Microbacterium sp. WCNS1B, (h) Azotobacter sp. WCNS3B, (i) Rhodococcus sp. WCNS2A, (j) Agromyces sp. WCNS1A, (k) Azotobacter sp. WCNS2B, (l) Rhodococcus sp. WCNS3C, (m) Arthrobacter sp. WCNS2E, (n) Azotobacter sp. WCNS1D, (o) Prescotella sp. WCNS2D, (p) Azotobacter sp. WCNS3F, (q) Azotobacter sp. WCNS3E.
Figure 2. Germination bioassay using H-311 maize hybrid seeds inoculated with: (a) Control, (b) Leifsonia sp. WCNS2G, (c) Agromyces sp. WCNS1F, (d) Rhodococcus sp. WCNS3A (e) Terrabacter sp. WCNS1C, (f), Sinomonas sp. WCNS2C, (g) Microbacterium sp. WCNS1B, (h) Azotobacter sp. WCNS3B, (i) Rhodococcus sp. WCNS2A, (j) Agromyces sp. WCNS1A, (k) Azotobacter sp. WCNS2B, (l) Rhodococcus sp. WCNS3C, (m) Arthrobacter sp. WCNS2E, (n) Azotobacter sp. WCNS1D, (o) Prescotella sp. WCNS2D, (p) Azotobacter sp. WCNS3F, (q) Azotobacter sp. WCNS3E.
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Figure 3. Principal Component Analysis (PCA) plot of the effect of the bacterial strains on the maize considering the following parameters: plumule length (PL), seedling root length (RL), seedling length (SL), number of roots (NR), plumule fresh weight (PFW), plumule dry weight (PDW), roots fresh weight (RFW), roots dry weight (RDW), seedling fresh weight (SFW), and seedling dry weight (SDW). The name of the strains was reduced to the last two characters.
Figure 3. Principal Component Analysis (PCA) plot of the effect of the bacterial strains on the maize considering the following parameters: plumule length (PL), seedling root length (RL), seedling length (SL), number of roots (NR), plumule fresh weight (PFW), plumule dry weight (PDW), roots fresh weight (RFW), roots dry weight (RDW), seedling fresh weight (SFW), and seedling dry weight (SDW). The name of the strains was reduced to the last two characters.
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Figure 4. Effect of tequila vinasse irrigated soils (S1, S2, S3) in the germination of maize seeds.
Figure 4. Effect of tequila vinasse irrigated soils (S1, S2, S3) in the germination of maize seeds.
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Figure 5. H-311 maize hybrid seeds inoculated with: (a,b) Terrabacter sp. WCNS1C; (c,d) Azotobacter sp. 1D, (e,f) Azotobacter sp. 2A; (g,h) Prescottella sp. 2D; (i,j) Leifsonia sp. WCNS2G; (k,l) Rhodococcus sp. WCNS3A; (m,n) Agromyces sp. WCNS1F; (o,p) Azotobacter sp. 3E; (q,r) Rhodococcus sp. 3C; (s) positive control, (t) negative control. Tequila vinasse was added to the seeds of blanks in (a,c,e,g,i,k,m,o,q) and the positive control (s).
Figure 5. H-311 maize hybrid seeds inoculated with: (a,b) Terrabacter sp. WCNS1C; (c,d) Azotobacter sp. 1D, (e,f) Azotobacter sp. 2A; (g,h) Prescottella sp. 2D; (i,j) Leifsonia sp. WCNS2G; (k,l) Rhodococcus sp. WCNS3A; (m,n) Agromyces sp. WCNS1F; (o,p) Azotobacter sp. 3E; (q,r) Rhodococcus sp. 3C; (s) positive control, (t) negative control. Tequila vinasse was added to the seeds of blanks in (a,c,e,g,i,k,m,o,q) and the positive control (s).
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Figure 6. Principal Component Analysis (PCA) plot of the effect of the bacterial strains and tequila vinasse on the maize seedlings, considering the parameters: plumule length (PL), seedling root length (RL), seedling length (SL), number of roots (NR), plumule fresh weight (PFW), plumule dry weight (PDW), roots fresh weight (RFW), roots dry weight (RDW), seedling fresh weight (SFW), and seedling dry weight (SDW). The name of the strains was reduced to the last two characters. Treatments indicated with V were added with tequila vinasse 1.25%, and treatments with H correspond to blanks. PC = positive control, NC = negative control.
Figure 6. Principal Component Analysis (PCA) plot of the effect of the bacterial strains and tequila vinasse on the maize seedlings, considering the parameters: plumule length (PL), seedling root length (RL), seedling length (SL), number of roots (NR), plumule fresh weight (PFW), plumule dry weight (PDW), roots fresh weight (RFW), roots dry weight (RDW), seedling fresh weight (SFW), and seedling dry weight (SDW). The name of the strains was reduced to the last two characters. Treatments indicated with V were added with tequila vinasse 1.25%, and treatments with H correspond to blanks. PC = positive control, NC = negative control.
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Table 1. Bacterial strains isolated from tequila vinasses irrigated soils.
Table 1. Bacterial strains isolated from tequila vinasses irrigated soils.
Isolation
Source		StrainUFC g-1 TVCSGenBank Accession
Number		TV-MICs (%)
WDW
Soil 1WCNS1A5.0 × 104OM06044015.07.5
(S1)WCNS1B5.0 × 104OM060441-7.5
WCNS1C1.6 × 105OM06044212.5>30.0
WCNS1D2.0 × 104OM06044317.5>30.0
WCNS1E5.1 × 105OM06044425.07.5
WCNS1F9.5 × 105OM06044512.57.5
Soil 2WCNS2A5.0 × 104OM060446>30.0>30.0
(S2)WCNS2B2.9 × 105OM06044720.07.5
WCNS2C1.6 × 105OM06044825.07.5
WCNS2D1.3 × 105OM06044925.0>30.0
WCNS2E4.2 × 105OM06045022.57.5
WCNS2F1.7 × 105OM06045120.07.5
WCNS2G1.0 × 105OM06045222.57.5
Soil 3WCNS3A4.0 × 104OM06045322.5>30.0
(S3)WCNS3B1.1 × 105OM060454>30.015.0
WCNS3C4.0 × 104OM06045520.030.0
WCNS3D1.0 × 104OM06045612.57.5
WCNS3E5.0 × 104OM060457>30.015.0
WCNS3F1.0 × 104OM06045815.07.5
Soil 1 = Vinasses poured continuously for one month, annually, for two years. Soil 2 = Vinasses poured continuously for two months, annually, over a span of ten years. Soil 3 = Vinasses poured continuously for one month, three to four times per year, over a period of fourteen years. TVCS = tequila vinasses irrigated soils, TV-MICs = Tequila vinasses minimal inhibitory concentrations, WD = Winogradsky medium with dextrose, W = Winogradsky medium without dextrose.
Table 2. Identity between the 16S rRNA of the bacterial strains form isolated from tequila vinasses irrigated soils and the most related bacterial type strain.
Table 2. Identity between the 16S rRNA of the bacterial strains form isolated from tequila vinasses irrigated soils and the most related bacterial type strain.
StrainSpecies IdentifiedIdentity (%)Related Type Strain
WCNS1AAgromyces sp.99.9Agromyces fucosus NR_104982
WCNS1BMicrobacterium sp.99.7Microbacterium liquefaciens NR_026162
WCNS1CTerrabacter sp.99.1Terrabacter terrae NR_043286
WCNS1DAzotobacter sp.97.6Azotobacter bryophylli MF078077
WCNS1EPseudomonas sp.99.6Pseudomonas harudinis NR_181730
WCNS1FAgromyces sp.99.9Agromyces fucosus NR_104982
WCNS2AAzotobacter sp.97.5Azotobacter bryophylli MF078077
WCNS2BAzotobacter sp.97.4Azotobacter bryophylli MF078077
WCNS2CSinomonas sp.99.5Sinomonas atrocyanea CP014518
WCNS2DPrescottella sp.100Prescottella equi X80614
WCNS2EArthrobacter sp.98.4Arthrobacter globiformi NR_112192
WCNS2FStreptomyces sp.100Streptomyces bungoensis NR_041191
WCNS2GLeifsonia sp.98.9Leifsonia xyli CP006734
WCNS3ARhodococcus sp.100Rhodococcus ruber OQ345823
WCNS3BAzotobacter sp.97.5Azotobacter bryophylli MF078077
WCNS3CRhodococcus sp.100Rhodococcus ruber OQ345823
WCNS3DAzotobacter sp.97.5Azotobacter bryophylli MF078077
WCNS3EAzotobacter sp.97.5Azotobacter bryophylli MF078077
WCNS3FAzotobacter sp.97.4Azotobacter bryophylli MF078077
Table 3. Plant growth promotion and biocontrol in vitro evaluation of bacterial strains isolated from tequila vinasse-irrigated soils.
Table 3. Plant growth promotion and biocontrol in vitro evaluation of bacterial strains isolated from tequila vinasse-irrigated soils.
Solubilization Halo (cm)Siderophore Halo (cm)Hydrolytic Enzyme Halo (cm)
StrainKPO4AmylaseProteaseCellulaseLipaseEsterase
WCNS1A----0.96 e1.82 a--
WCNS1B-----0.35 l--
WCNS1C----0.07 op1.06 d--
WCNS1D--------
WCNS1E0.10 o0.56 j0.87 f--1.37 b--
WCNS1F0.37 l-0.24 m---1.23 c1.05 d
WCNS2A--------
WCNS2B--------
WCNS2C------0.84 fg-
WCNS2D--------
WCNS2E-----0.64 i--
WCNS2F--------
WCNS2G--0.09 o-----
WCNS3A--0.07 op----0.81 g
WCNS3B--------
WCNS3C--0.17 n0.04 p---0.46 k
WCNS3D--------
WCNS3E--0.73 h-----
WCNS3F--------
Values followed by the same letter(s) represent no significant differences noted from Tukey’s test (p ≤ 0.05), (-) = no halo was generated.
Table 4. Maize seedling growth-promoting test of seeds inoculated with strains from tequila vinasse-irrigated soils.
Table 4. Maize seedling growth-promoting test of seeds inoculated with strains from tequila vinasse-irrigated soils.
StrainSeedling Parameters
PL
(cm)		RL
(cm)		SL
(cm)		NRRFW
(g)		RDW
(g)		PFW
(g)		PDW
(g)		SFW
(g)		SDW
(g)
WCNS1A20.8 abc19.0 a39.8 ab4.2 abc0.49 abcd0.12 ab3.14 c0.48 abc3.6 bc0.60 abc
WCNS1B19.7 abcd11.8 c31.4 de4.0 abc0.43 cdef0.09 bcde2.79 e0.45 bc3.2 def0.54 bc
WCNS1C22.0 ab19.3 a41.3 ab4.5 abc0.56 a0.13 a3.06 d0.54 ab3.6 b0.67 ab
WCNS1D20.6 abcd20.9 a41.5 ab5.3 a0.41 def0.10 bcd2.45 fg0.48 abc2.9 gh0.58 abc
WCNS1F20.7 abcd19.3 a40.0 ab4.3 abc0.45 bcd0.12 ab3.14 c0.59 a3.6 bc0.71 a
WCNS2A22.3 ab20.8 a43.1 a4.2 abc0.44 bcde0.12 ab3.65 a0.52 ab4.1 a0.64 abc
WCNS2B20.0 abcd18.6 a38.6 abc3.8 abc0.49 abcd0.09 cde2.76 e0.44 bc3.3 de0.53 c
WCNS2C19.8 abcd17.8 ab37.6 abc5.0 ab0.29 g0.09 bcde2.90 de0.48 abc3.2 def0.67 ab
WCNS2D19.6 abcd17.5 ab37.1 bcd4.2 abc0.55 a0.14 a2.74 e0.40 c3.3 de0.54 bc
WCNS2E16.4 d12.7 c29.2 e3.7 bc0.36 efg0.06 e2.73 e0.48 abc3.1 efg0.55 bc
WCNS2G23.3 a14.3 bc37.7 abc5.3 a0.52 ab0.09 cde3.66 a0.58 abc4.2 a0.67 abc
WCNS3A20.9 abc19.5 a40.4 ab3.5 bc0.42 def0.11 abcd3.37 b0.54 ab3.8 b0.65 abc
WCNS3B23.1 ab18.2 ab41.3 ab3.8 abc0.51 abc0.09 bcde3.65 b0.50 abc4.2 a0.59 abc
WCNS3C20.0 abcd18.2 ab38.2 abc3.5 bc0.46 bcd0.10 bcd2.90 de0.49 abc3.4 cd0.59 abc
WCNS3E19.8 abcd18.5 ab38.3 abc3.8 abc0.49 abcd0.10 bcd2.28 g0.48 abc2.8 h0.58 abc
WCNS3F18.9 bcd14.3 bc33.2 cde4.0 abc0.45 bcde0.12 abc2.52 f0.45 bc3.0 fgh0.57 bc
C17.6 cd17.3 ab36.5 bcd3.2 c0.35 fg0.08 de2.75 e0.47 abc3.1 efg0.56 bc
PL = plumule length, RL = seedling root length, SL = seedling length, NR = number of roots, RFW = roots fresh weight, RDW = roots dry weight, PFW = plumule fresh weight, PDW = plumule dry weight, SFW = seedling fresh weight, SDW = seedling dry weight. The superscript lowercase letters indicate the groups according to Tukey’s test.
Table 5. Maize seedling growth-promoting test of seeds inoculated with strains from tequila vinasse-irrigated soils with 2% neutralized vinasses.
Table 5. Maize seedling growth-promoting test of seeds inoculated with strains from tequila vinasse-irrigated soils with 2% neutralized vinasses.
StrainSeedling Parameters
PL
(cm)		RL
(cm)		SL
(cm)		NRRFW
(g)		RDW
(g)		PFW
(g)		PDW
(g)		SFW
(g)		SDW
(g)
WCNS1C-V24.2 abc23.6 abc47.8 ab5.3 a0.61 abc0.13 abc4.7 ab0.71 abc5.3 abc0.85 abc
WCNS1C-H21.3 cdef19.8 abc41.2 bcdef4.4 abc0.57 bcde0.13 abc3.0 e0.58 defgh3.6 g0.71 efghi
WCNS1D-V24.7 ab21.9 ab46.5 abc5.7 a0.70 a0.13 abc4.6 b0.73 ab5.3 bcd0.85 ab
WCNS1D-H21.3 cdef21.4 cdef42.7 abcde4.8 abc0.39 gh0.09 c2.7 e0.50 ghi3.1 gh0.60 j
WCNS1F-V21.8 abcdef24.0 abcdef45.8 abc5.3 ab0.57 bcde0.15 a4.3 bcd0.68 abcd4.9 bcdef0.83 abcd
WCNS1F-H21.6 bcdef19.3 bcdef40.9 bcdef4.4 abc0.48 defg0.12 abc3.1 e0.51 ghi3.6 g0.63 hij
WCNS2A-V23.0 abcde24.8 abcde47.8 ab5.2 ab0.72 a0.15 a4.5 bc0.75 a5.2 abcd0.90 a
WCNS2A-H20.4 efg21.4 efg41.8 bcde4.2 abc0.47 efg0.12 abc3.8 d0.55 efghi4.3 f0.68 efghij
WCNS2D-V21.1 abcde22.5 abcde43.6 abcde5.5 a0.56 cde0.15 a4.2 bcd0.64 bcde4.7 cdef0.79 cde
WCNS2D-H19.9 efg16.7 efg36.6 ef3.9 abc0.49 defg0.11 abc3.8 d0.55 efghi4.3 f0.66 fghij
WCNS2G-V22.7 abcde21.5 abcde44.1 abcd5.0 ab0.55 cdef0.13 abc5.2 a0.63 cdef5.8 a0.76 bcdef
WCNS2G-H21.6 bcdef20.9 bcdef42.5 bcde4.5 abc0.50 defg0.12 abc4.5 bc0.54 efghi5.0 bcde0.66 fghij
WCNS3A-V23.9 abcd23.9 abcd47.7 ab5.1 ab0.66 a0.14 ab4.7 ab0.58 defgh5.3 ab0.72 defghi
WCNS3A-H21.8 abcdef22.1 abcdef43.9 abcde3.9 abc0.50 defg0.13 abc3.0 e0.55 efghi3.5 g0.68 efghij
WCNS3C-V25.0 a25.0 a49.9 a4.8 abc0.49 defg0.14 ab3.9 cd0.60 cdfg4.4 def0.74 cdefgh
WCNS3C-H19.2 fg20.9 fg40.1 cdef3.1 c0.41 fgh0.11 abc2.8 e0.47 i3.2 gh0.58 j
WCNS3E-V22.1 abcdef21.5 abcdef43.5 abcde4.4 abc0.66 abc0.12 abc3.8 d0.62 cdef4.4 ef0.74 cdefg
WCNS3E-H20.6 defg18.8 defg39.4 cdef3.8 abc0.48 defg0.10 bc2.8 e0.53 fghi3.3 gh0.63 ghij
PC19.0 fg19.0 fg38.0 def3.8 abc0.38 gh0.10 bc2.9 e0.51 ghi3.2 gh0.61 ij
NC18.0 g16.3 g34.2 f3.4 bc0.31 h0.10 bc2.5 e0.48 hi2.9 h0.58 j
p value of significance
Treat<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001
Strain<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001<0.001
Treat × Strain0.0010.3270.2480.735<0.0010.094<0.001<0.001<0.001<0.001
PL = plumule length, LR = seedling root length, SL = seedling length, NR = number of roots, RFW = roots fresh weight, RDW = roots dry weight, PFW = plumule fresh weight, PDW = plumule dry weight, SFW = seedling fresh weight, SDW = seedling dry weight. Treat = Treatments, Strain = bacterial strains. PC = positive control, NC = negative control. Strains indicated with “-V” correspond to treatments evaluating their plant-promoting capacities in the presence of tequila vinasse (1.25%). Strains indicated with “-H” correspond to blanks. The superscript lowercase letters indicate the groups according to Tukey’s test.
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Valencia-Botín, A.J.; Chávez-Díaz, I.F.; Zurita-Martínez, F.; Tejeda-Ortega, A.; Zelaya-Molina, L.X. Plant Growth-Promoting and Tequila Vinasse-Resistant Bacterial Strains. Microbiol. Res. 2024, 15, 1144-1162. https://doi.org/10.3390/microbiolres15030077

AMA Style

Valencia-Botín AJ, Chávez-Díaz IF, Zurita-Martínez F, Tejeda-Ortega A, Zelaya-Molina LX. Plant Growth-Promoting and Tequila Vinasse-Resistant Bacterial Strains. Microbiology Research. 2024; 15(3):1144-1162. https://doi.org/10.3390/microbiolres15030077

Chicago/Turabian Style

Valencia-Botín, Alberto J., Ismael F. Chávez-Díaz, Florentina Zurita-Martínez, Allan Tejeda-Ortega, and Lily X. Zelaya-Molina. 2024. "Plant Growth-Promoting and Tequila Vinasse-Resistant Bacterial Strains" Microbiology Research 15, no. 3: 1144-1162. https://doi.org/10.3390/microbiolres15030077

APA Style

Valencia-Botín, A. J., Chávez-Díaz, I. F., Zurita-Martínez, F., Tejeda-Ortega, A., & Zelaya-Molina, L. X. (2024). Plant Growth-Promoting and Tequila Vinasse-Resistant Bacterial Strains. Microbiology Research, 15(3), 1144-1162. https://doi.org/10.3390/microbiolres15030077

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